JP5559529B2 - Fluorescent probe - Google Patents

Description

  The present invention relates to a fluorescent probe. More specifically, the present invention relates to a fluorescent probe using fluorescence resonance energy transfer that can use excitation light / fluorescence in the near infrared region.

  In recent years, fluorescent probes for imaging biological phenomena have been actively developed in biochemical research, and various fluorescent probes for pH, various metal ions, reactive oxygen species, and the like have been proposed. For example, BCECF (2 ′, 7′-bis (carboxyethyl) -4 or 5-carboxyfluorescein) and its derivatives, CFDA (carboxyfluorescein diacetate) and its derivatives, SNARF-1 (seminaphthorhodafluor) and its fluorescent probe for measuring pH Derivatives thereof [both Chapter 20 (pH indicator) of the catalog of Molecular Probes (Handbook of Fluorescent Probes and Research Chemicals, Tenth edition)] have been proposed. Moreover, TSQ (Reyes, JG, et al., Biol. Res., 27, 49, 1994), Zinquin ethyl ester (Tsuda, M., et al., Neurosci., As a fluorescent probe for measuring zinc ions. 17, 6678, 1997), Dansylaminoethylcyclen (Koike, T., et al., J. Am. Chem. Soc., 118, 12686, 1996), Newport Green [Handbook of Fluorescent Probes and Research Chemicals, Tenth edition), Chapter 19 (Calcium ions, Magnesium ions, Zinc ions, and other metal ions)], and further by the present inventors, a zinc ion probe having fluorescein as a fluorescent host nucleus (Japanese Patent Laid-Open No. 2000-239272). No. 1, International Publication No. WO01 / 062775, and Japanese Unexamined Patent Publication No. 2004-315501) and a zinc ion probe using benzofuran as a fluorescent host nucleus (International Publication No. WO02 / 102795) have been proposed.

  These fluorescent probes desirably change in fluorescence characteristics only when they react with a target measurement object, but are often affected by other factors. In particular, when applying fluorescent probes to cells or biological tissues, the concentration of fluorescent probes introduced by cells varies, the intensity of excitation light at the measurement site varies depending on the thickness of the cell membrane, and hydrophobicity of the membrane There is a problem that there are many factors that affect the measurement, such as the possibility that the fluorescent probe is localized at a high portion.

  The ratio measurement method has been developed and used as a method for reducing the measurement error due to these factors and performing accurate quantitative analysis (Kawanishi Y., et al, Angew. Chem. Int. Ed., 39 (19), 3438, 2000). This method includes a step of measuring the fluorescence intensity at two different wavelengths in the fluorescence spectrum or the excitation spectrum and detecting the ratio, and the influence of the concentration of the fluorescent probe itself and the excitation light intensity can be ignored. It is possible to eliminate measurement errors due to the localization, concentration change, or fading of the fluorescent probe itself that occurs when observation is performed at a wavelength. In order to enable such ratio measurement, a fluorescent probe whose excitation light wavelength or fluorescence wavelength changes before and after the reaction with the measurement object is required. For example, the pH fluorescent probe SNARF-1 has the property that when the pH is biased to alkalinity, the peak of the fluorescence wavelength shifts to the long wavelength side due to deprotonation, and when excited near 500 nm, the fluorescence near 580 nm The intensity decreases with increasing pH, while the fluorescence intensity near 640 nm increases with increasing pH. Therefore, by irradiating the solution containing this compound with excitation light, measuring the fluorescence intensity of the appropriate two wavelengths at that time, and determining the ratio, it is possible regardless of the probe concentration, light source intensity, cell size, etc. Can measure pH accurately. Moreover, the zinc ion fluorescent probe described in International Publication No. WO02 / 102795 is a fluorescent probe based on the principle of intramolecular charge transfer, and when the zinc ion is not captured, the excitation wavelength is 354 nm and the fluorescence wavelength is 532 nm. However, depending on the concentration of zinc ions, the peak of the excitation spectrum is blue-shifted by about 20 nm.For example, using 335 nm and 354 nm as the excitation wavelength, the fluorescence intensity at each excitation wavelength is measured and the ratio is calculated. As a result, the zinc ion concentration can be accurately measured regardless of the probe concentration, light source intensity, cell size, and the like. However, these are ratio type fluorescent probes that use an excitation light wavelength / fluorescence wavelength of a wavelength below the visible light region, and there is a problem that such excitation light has low permeability of living tissue. Furthermore, the problem of being easily affected by the autofluorescence of the cell system itself during measurement has not been solved.

  On the other hand, cyanine dyes are widely used in various fields, and are also used as fluorescent labels for biomolecules in the fluorescence imaging region for studying physiological functions. Among these cyanine dyes, tricarbocyanine dyes are fluorescent dyes having a maximum absorption wavelength and a maximum fluorescence wavelength in the near-infrared region near 650 nm to 950 nm, which are relatively less absorbed by biomolecules. This has the advantage that light having a wavelength that can be transmitted up to is usable. In addition, since the near-infrared region has little autofluorescence from biological components, the characteristics of tricarbocyanine dyes are suitable for in vivo imaging.

  Recently, in addition to cyanine dyes for direct fluorescent labeling of biomolecules, tricarbocyanine dyes whose fluorescence intensity changes by reacting specifically with biomolecules have been developed. For example, near-infrared fluorescent probes for calcium ions (Ozmen, B., et al., Tetrahedron Lett., 41, pp.9185-9188, 2000), near-infrared fluorescent probes for nitric oxide (NO) (publicly available) WO2005 / 080331). These fluorescent probes are probes in which only the fluorescence intensity changes without the excitation / fluorescence wavelength changing before and after a specific reaction with a biomolecule. Examples of pH probes include compounds described in International Publication No. WO00 / 075237 and CypHer (GE Healthcare Bioscience Co., Ltd.). These have the principle that the nitrogen atom of the nitrogen-containing heteroaromatic ring bonded to the polymethine chain of the cyanine skeleton is protonated to emit fluorescence, and the fluorescence intensity increases below the neutral region as the pH decreases. Furthermore, the present inventors recently developed a near-infrared fluorescent probe for zinc ions (Japanese Patent Application No. 2005-057265) and a near-infrared fluorescent probe used for pH measurement (Japanese Patent Application No. 2007-035768). These are ratio fluorescent probes whose excitation wavelength shifts according to changes in pH and zinc ion concentration, have a maximum absorption wavelength between 650 nm and 800 nm, which excels in biopermeability, and an excitation spectrum depending on zinc ion concentration or pH change. In order to produce a remarkable wavelength shift in the peak of the light, it is very accurate to irradiate the living body by irradiating excitation light of appropriate two wavelengths of 650 nm to 800 nm, measuring the fluorescence intensity at each excitation wavelength, and determining the ratio. Zinc ion concentration and pH change in deep tissue can be measured.

  In addition, since the fluorescence change due to fluorescence resonance energy transfer (FRET) is also a two-wavelength ratio type, ratio measurement is possible even with FRET type fluorescent probes, regardless of probe concentration, light source intensity, cell size, etc. It can be measured quantitatively. However, most of the conventionally known fluorescent probes using FRET (for example, the fluorescent probes described in Japanese Patent Application Laid-Open No. 2000-316598) use fluorescence in the visible light region and excite in the near infrared region. As a fluorescent probe using the light wavelength / fluorescence wavelength, there is little known other than the description of the FRET type pH probe using on / off of fluorescence accompanying pH change in International Publication No. WO00 / 075237. In addition, the probe described in International Publication No. WO00 / 075237 can only be used as a pH probe, and has a problem that it cannot be applied as a probe for other substances to be measured such as metal ions and enzyme substrates.

  An object of the present invention is to provide a fluorescent probe in which the excitation light wavelength / fluorescence wavelength is in the near infrared region excellent in biological tissue permeability and can measure a measurement target substance by the ratio method. In particular, the object is to provide a pH fluorescent probe containing a compound having the above characteristics, a pH measuring method using the pH fluorescent probe, a zinc ion fluorescent probe, and a zinc ion measuring method using the zinc ion fluorescent probe.

  As a result of diligent efforts to solve the above-mentioned problems, the present inventors have an excitation wavelength / fluorescence wavelength in the near-infrared region, and the excitation light wavelength greatly changes by specifically reacting with the measurement target substance in the sample. Specific fluorescent compound (fluorophore A to be described later) and a compound which itself has fluorescence in the near infrared region and functions as a donor or acceptor having a FRET relationship with the specific fluorescent compound It has been found that by using a fluorescent probe linked with (fluorophore B described later), the reaction between the fluorescent probe and a substance to be measured can be measured using fluorescence as a change in FRET efficiency of the fluorescent probe. In addition, by using this method, the present inventors can measure a measurement target substance in a sample by a ratio method using an excitation wavelength / fluorescence wavelength in the near-infrared region having excellent biological tissue permeability. The present inventors have found that the measurement target substance in the deep part of the living tissue can be measured very accurately without being affected by the difference in the environment of the living tissue or the autofluorescence derived from the endogenous substance in the living tissue. The present invention has been completed based on these findings.

［式中、R¹及びR²はそれぞれ独立に置換基を有していてもよいC_1-18アルキル基を示し；R³及びR⁴はそれぞれ独立に水素原子を示すか、又は測定対象物質を検出するための置換基を示すが、R³及びR⁴が同時に水素原子であることはなく、R³及びR⁴が互いに結合して測定対象物質を検出するための環構造を形成してもよく；Y¹及びY²はそれぞれ独立に-O-、-S-、-Se-、-CH=CH-、-C(R⁵)(R⁶)-、又は-N(R⁷)-（式中、R⁵、R⁶、及びR⁷はそれぞれ独立に水素原子、又は置換基を有していてもよいC_1-6アルキル基を示す）を示し；n及びn'はそれぞれ独立に0、1、又は2を示し；Z¹及びZ²はそれぞれ独立に置換基を有していてもよいベンゾ縮合環又は置換基を有していてもよいナフト縮合環を形成するために必要な非金属原子群を示し；L¹、L²、L³、L⁴、L⁵、L⁶、及びL⁷はそれぞれ独立に置換又は無置換のメチン基を示すが、n又はn'が2である場合には重複するL¹及びL²又は重複するL⁶とL⁷は同一でも異なっていてもよく、；M^-は電荷の中和に必要な個数の対イオンを示し、ただしR³及びR⁴の組み合わせは、測定対象物質の検出の前後で、式(AI)で表される蛍光団Aの極大励起波長が変化する組合せである］で表される蛍光プローブが提供される。That is, according to the present invention, the following general formula (I):
Fluorophore A-S-Fluorophore B (I)
(However, fluorophore A and fluorophore B are fluorophores that emit fluorescence when irradiated with excitation light having a wavelength of 600 nm to 950 nm, respectively, and fluorophore A has a specific reaction with the analyte. Fluorescence properties change before and after, and S is a spacer that connects fluorophore A and fluorophore B), and fluorophore A and fluorescence before and after specific reaction with the analyte A fluorescent probe comprising a compound that causes a substantial change in the efficiency of fluorescence resonance energy transfer with the group B, wherein the group A has the following formula (AI):

[Wherein, R ¹ and R ² each independently represent a C _1-18 alkyl group _optionally having a substituent; R ³ and R ⁴ each independently represent a hydrogen atom, or a substance to be measured R ³ and R ⁴ are not hydrogen atoms at the same time, and R ³ and R ⁴ are bonded to each other to form a ring structure for detecting the measurement target substance. Y ¹ and Y ² are each independently —O—, —S—, —Se—, —CH═CH—, —C (R ⁵ ) (R ⁶ ) —, or —N (R ⁷ ) —. (Wherein R ⁵ , R ⁶ and R ⁷ each independently represents a hydrogen atom or an optionally substituted C _1-6 alkyl group); and n and n ′ each independently 0, 1, or 2; each of Z ¹ and Z ² is necessary to form a benzo-fused ring which may have a substituent or a naphth-fused ring which may have a substituent. shows a non-metallic atomic ^{group; L 1, L 2, L} 3, L 4, L 5, L 6 And L ⁷ are each independently represent a substituted or unsubstituted methine group, L ⁶ and L ⁷ to L ¹ and L ² or overlapping overlapping when n or n 'is 2 be the same or different M ⁻ represents the number of counter ions required for charge neutralization, provided that the combination of R ³ and R ⁴ is a fluorophore represented by the formula (AI) before and after detection of the analyte. Is a combination in which the maximum excitation wavelength of A is changed].

［式中、R¹¹及びR¹²はそれぞれ独立に置換基を有していてもよいC_1-18アルキル基を示し；R¹³及びR¹⁴はそれぞれ独立に水素原子を示すか、又は測定対象物質を検出するための置換基を示すが、R¹³及びR¹⁴が同時に水素原子であることはなく、R¹³及びR¹⁴が互いに結合して測定対象物質を検出するための環構造を形成してもよく；Y¹¹及びY¹²はそれぞれ独立に-O-、-S-、-Se-、-CH=CH-、-C(R¹⁵) (R¹⁶)-、又は-N(R¹⁷)-（式中、R¹⁵、R¹⁶、及びR¹⁷はそれぞれ独立に水素原子又は置換基を有していてもよいC_1-6アルキル基を示す）を示し；Z¹¹及びZ¹²はそれぞれ独立に置換基を有していてもよいベンゾ縮合環又は置換基を有していてもよいナフト縮合環を形成するために必要な非金属原子群を表し；M'^-は電荷の中和に必要な個数の対イオンを示し；ただしR¹³及びR¹⁴の組み合わせは、測定対象物質の検出の前後で式(AII)で表される蛍光団Aの極大励起波長が変化する組合せである］で表される蛍光プローブが提供される。According to a preferred embodiment of the invention, the fluorophore A in the general formula (I) is represented by the following formula (AII):

[Wherein, R ¹¹ and R ¹² each independently represent a C _1-18 alkyl group _optionally having a substituent; R ¹³ and R ¹⁴ each independently represent a hydrogen atom, or a substance to be measured R ¹³ and R ¹⁴ are not hydrogen atoms at the same time, and R ¹³ and R ¹⁴ are bonded to each other to form a ring structure for detecting the measurement target substance. Y ¹¹ and Y ¹² are each independently —O—, —S—, —Se—, —CH═CH—, —C (R ¹⁵ ) (R ¹⁶ ) —, or —N (R ¹⁷ ) —. Wherein R ¹⁵ , R ¹⁶ and R ¹⁷ each independently represents a hydrogen atom or a C _1-6 alkyl group which may have a substituent; and Z ¹¹ and Z ¹² each independently Represents a group of non-metallic atoms necessary to form an optionally substituted benzo-fused ring or an optionally substituted naphtho-fused ring; M ′ ⁻ is necessary for charge neutralization It represents a counter ion in a number proviso R ¹³及The combination of R ¹⁴ is a fluorescent probe that maximum excitation wavelength of the fluorophores A represented by formula (AII) before and after the detection of the analyte is represented by a Combination of change is provided.

  According to a further preferred aspect of the present invention, the fluorescent probe wherein the substance to be measured is a proton, metal ion, reactive oxygen species, or enzyme reaction substrate or enzyme reaction product; the fluorophore B is tetramethylindodicarbocyanine The above-described fluorescent probe, which is a derivative and the fluorophore A is represented by the above formula (AII); the above-described fluorescent probe in which the metal ion is an alkali metal ion, calcium ion, magnesium ion, or zinc ion; The above fluorescent probe selected from the group consisting of nitric oxide, hydroxy radical, singlet oxygen, hydrogen peroxide, peroxynitrite, hypochlorite ion, and superoxide; and the enzyme is a reductase, oxidase, and The above-described fluorescent probe selected from the group consisting of hydrolase is provided.

According to another preferred embodiment, the fluorescent probe used for pH measurement; the fluorescent probe used for pH measurement, wherein the fluorophore A is represented by the above formula (AI) (wherein R ¹ and R ² are each independently A methyl group or a 4-sulfobutyl group; either one of R ³ and R ⁴ is a hydrogen atom and the other is an optionally substituted C _1-18 alkyl group; Y ¹ and Y ² is —C (CH ₃ ) ₂ —; n and n ′ are both 1; Z ¹ and Z ² are both unsubstituted benzo-fused rings; L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , and L ⁷ are unsubstituted methine groups; M ⁻ represents the number of counter ions necessary for charge neutralization), and fluorophore A is represented by the above formula ( AI) (wherein R ¹ and R ² are each independently a methyl group or a 4-sulfobutyl group; R ³ and R ⁴ are each independently a C _1-18 alkyl group which may have a substituent, a substituent Or an aryl group optionally having-or-(CH ₂ ) _m --N (R ²¹ ) (R ²² ), and at least one of R ³ and R ⁴ is-(CH ₂ ) _m --N (R ²¹ ) (R ²² ) (R ²¹ and R ²² each independently represents a C _1-6 alkyl group which may have a substituent or an aryl group which may have a substituent, or R ²¹ or R ²² represents a C _1-3 alkylene group which may have a substituent bonded to R ³ or R ⁴ respectively, and m represents 1 or 2); Y ¹ and Y ² represent —C (CH ₃ ) ₂ —; n and n ′ are both 1, Z ¹ and Z ² are unsubstituted benzo-fused rings, and L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ And L ⁷ is an unsubstituted methine group; M ⁻ represents the number of counter ions necessary for charge neutralization).

As the fluorescent probe for measuring pH, a fluorescent probe containing the following compound is particularly preferable.

（式中、W¹、W ²、W ³、及びW ⁴はそれぞれ独立に水素原子、2-ピリジルメチル基、2-ピリジルエチル基、2-メチル-6-ピリジルメチル基、又は2-メチル-6-ピリジルエチル基を示すが、W¹、W ²、W ³、及びW ⁴からなる群から選ばれる基のうち少なくとも１つは2-ピリジルメチル基、2-ピリジルエチル基、2-メチル-6-ピリジルメチル基、及び2-メチル-6-ピリジルエチル基からなる群から選ばれる基を示し；p及びｑはそれぞれ独立に0又は1を示す）で表される基であり（ただし、R³及びR⁴がともに水素原子であることはなく）；Y¹及びY²が-C(CH₃)₂-であり；n及びn'がともに1であり；Z¹及びZ²が無置換ベンゾ縮合環であり；L¹、L²、L³、L⁴、L⁵、L⁶、及びL⁷が無置換のメチン基であり；M⁻は電荷の中和に必要な個数の対イオンを示す）で表される蛍光プローブが提供される。From another aspect, according to the present invention, there is provided a fluorescent probe for measuring zinc ions containing a compound represented by the above general formula (I), wherein the fluorophore A is represented by the above formula (AI) [wherein R ¹ and R ² are each independently a methyl group or a 4-sulfobutyl group; R ³ and R ⁴ are each independently a hydrogen atom, or the following formula (D):

(Wherein W ¹ , W ² , W ³ and W ⁴ are each independently a hydrogen atom, 2-pyridylmethyl group, 2-pyridylethyl group, 2-methyl-6-pyridylmethyl group, or 2-methyl- 6-pyridylethyl group, at least one of the groups selected from the group consisting of W ¹ , W ² , W ³ and W ⁴ is 2-pyridylmethyl group, 2-pyridylethyl group, 2-methyl- A group selected from the group consisting of a 6-pyridylmethyl group and a 2-methyl-6-pyridylethyl group; p and q each independently represents 0 or 1) (provided that R ³ and R ⁴ are not both hydrogen atoms); Y ¹ and Y ² are —C (CH ₃ ) ₂ —; n and n ′ are both 1; Z ¹ and Z ² are unsubstituted A benzo-fused ring; L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , and L ⁷ are unsubstituted methine groups; M ⁻ is the number of counter ions required for charge neutralization Fluorescence represented by Robe is provided.

  From another point of view, according to the present invention, a compound represented by the above general formula (I), preferably a compound represented by the above general formula (I) is converted into a fluorescent probe for measuring a substance to be measured, preferably pH. A method for use as a fluorescent probe or a zinc ion fluorescent probe; use of the compound represented by the general formula (I) for the production of the fluorescent probe; a method for measuring the fluorescence of a substance to be measured, comprising the following steps: ) A step of bringing the compound represented by the general formula (I) into contact with the substance to be measured; and (b) measuring the fluorescence intensity of the compound represented by the general formula (I) after the contact in the step (a). Thus, there is provided a method including a step of detecting a change in FRET efficiency due to contact with a measurement target substance.

  From another viewpoint, the method for producing a fluorescent probe comprising a compound represented by the above formula (I), which specifically reacts with a measurement target substance in a sample represented by the above formula (AI). Fluorophore A, whose fluorescence characteristics change, and fluorophore B, which is a donor or acceptor capable of inducing fluorescence resonance energy transfer between fluorophore A, are linked by a spacer, and the above formula (I A method comprising the step of producing a compound represented by) is also provided by the present invention.

  According to the present invention, there is provided a fluorescent probe capable of measuring a measurement target substance in a sample by a ratio method using an excitation wavelength / fluorescence wavelength in the near-infrared region excellent in biological tissue permeability. The compound represented by the above general formula (I) contained in the fluorescent probe of the present invention has the property that the fluorophore A specifically reacts with the substance to be measured and the maximum excitation wavelength changes greatly. This is because the electron donating ability of the nitrogen atom bonded to the methine chain in the cyanine fluorophore of the above formula (AI), preferably (AII), which is the fluorophore A is changed. That is, by selecting various groups that change the electron donating ability of the nitrogen atom to the cyanine fluorophore by a specific reaction with the measurement target substance, and performing fluorescence measurement before and after the reaction with the measurement target substance, the above general formula By detecting the change in fluorescence resonance energy transfer efficiency between the fluorophore A and the fluorophore B in (I), it becomes possible to measure various substances to be measured in deep tissues in the living body.

It is the figure which showed the result of having measured the change of the absorption spectrum and the fluorescence spectrum of the compound 5 under various pH. In the figure, (A) shows an absorption spectrum, (B) shows a fluorescence spectrum (excitation wavelength 640 nm), and (C) shows a fluorescence intensity ratio (excitation wavelength 640 nm) between 660 nm and 750 nm. All samples were measured in a 1: 1 mixed solvent of 100 mM phosphate buffer and acetonitrile (but containing 0.1% or less DMSO as an auxiliary solvent).

  The fluorescent probe represented by the above general formula (I) provided by the present invention substantially changes the FRET efficiency between the fluorophore A and the fluorophore B before and after the specific reaction with the measurement target substance. And is used as a fluorescent probe for measuring a measurement target substance by detecting a change in FRET efficiency caused by contact with the test target substance. The type of the substance to be measured is not particularly limited, and hydrogen ions, hydroxide ions, enzyme reaction substrates or enzyme reaction products, metal ions (for example, alkali metal ions such as sodium ion and lithium ion, alkaline earth such as calcium ion) Metal ions, magnesium ions, zinc ions, etc.), non-metal ions (such as carbonate ions), reactive oxygen species (eg, nitric oxide, hydroxy radical, singlet oxygen, hydrogen peroxide, peroxynitrite, hypochlorous acid) Ions, superoxide, etc.), but is not limited thereto. Examples of enzymes involved in the enzymatic reaction include β-lactamase, cytochrome P450 oxidase, β-galactosidase, β-glucosidase, β-glucuronidase, β-hexosaminidase, lactase, alkaline phosphatase and other reductases, oxidases , Hydrolase and the like can be mentioned, but are not limited thereto.

  In this specification, “FRET” means that one of two fluorophores (donor) when two fluorophores are present at close distances (within about 100 mm) in the molecule of one compound. ) And the excitation spectrum of the other (acceptor) are overlapped, irradiation with light of the excitation wavelength of the donor attenuates the fluorescence of the donor, which should be observed, and accepts it instead. A phenomenon in which body fluorescence is observed.

  In the present specification, when saying “may have a substituent” for a certain functional group, the type, number, and substitution position of the substituent are not particularly limited. For example, an alkyl group, an alkoxy group, An aryl group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom or an iodine atom), a hydroxy group, an amino group, a carboxy group or an ester thereof, or a sulfo group or an ester thereof as a substituent. May be. In the present specification, the aryl group may be a monocyclic or polycyclic aryl group, or a monocyclic or polycyclic heteroaryl group, but is preferably monocyclic or polycyclic. A cyclic aryl group, more preferably a phenyl group, can be used.

In the formula (AI), R ¹ and R ² each independently represent a C _1-18 alkyl group which may have a substituent. As the alkyl group, a linear, branched, cyclic, or a combination thereof can be used, for example, a C _1-15 alkyl group is preferable, a C _1-10 alkyl group is more preferable, More preferred is a C _1-6 alkyl group. For example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, 2-methylbutyl group, 1-methylbutyl Group, neopentyl group, 1,2-dimethylpropyl group, 1-ethylpropyl group, n-hexyl group, 4-methylpentyl group, 3-methylpentyl group, 2-methylpentyl group, 1-methylpentyl group, 3, 3-dimethylbutyl group, 2,2-dimethylbutyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,3-dimethylbutyl group, 2-ethylbutyl group 1-ethylbutyl group, 1-ethyl-1-methylpropyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, n-undecyl group, n-dodecyl group, n-tridecyl group , N-tetradecyl group, n-pentadecyl group, cyclopropyl group, cyclobuty Group, cyclopentyl group, cyclohexyl group, cycloheptyl group, cyclooctyl group, cyclopropylmethyl group, 1-cyclopropylethyl group, 2-cyclopropylethyl group, 3-cyclopropylpropyl group, 4-cyclopropylbutyl group, 5 -Cyclopropylpentyl group, 6-cyclopropylhexyl group, cyclobutylmethyl group, cyclopentylmethyl group, cyclobutylmethyl group, cyclopentylmethyl group, cyclohexylmethyl group, cyclohexylpropyl group, cyclohexylbutyl group, cycloheptylmethyl group, cyclooctyl Examples thereof include, but are not limited to, a methyl group and a 6-cyclooctylhexyl group. The alkyl group represented by R ¹ and R ² is preferably a linear alkyl group.

Examples of the substituent that can be present on the C _1-18 alkyl group represented by R ¹ and R ² include an alkoxy group, an aryl group, and a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom). ), A hydroxy group, an amino group, a carboxy group or an ester thereof, a sulfo group or an ester thereof, or a phospho group or an ester thereof. Among these, a carboxy group or a sulfo group is preferable, and an effect of significantly increasing the water solubility of the compound of the present invention can be obtained. For example, specific examples of the alkyl group having a substituent include a hydroxymethyl group, a 1-hydroxyethyl group, a 2-hydroxylethyl group, a 2-hydroxypropyl group, a 3-hydroxypropyl group, and a 4-hydroxybutyl group. Carboxymethyl group, sulfomethyl group, 2-sulfoethyl group, 3-sulfopropyl group, 4-sulfobutyl group and the like. May both R ¹ and R ² are a C _1-18 alkyl group unsubstituted or C _1-18 alkyl group or one of them may have a substituent. Preferably R ¹ and R ² is C _1-6 alkyl group substituted by straight-chain C _1-6 alkyl group or a sulfonic acid group, particularly preferably a methyl group. R ¹¹ and R ¹² in Formula (AII) are the same as R ¹ and R ² in Formula (AI) above.

In the formula (AI), R ³ and R ⁴ each independently represent a hydrogen atom or a substituent for detecting the measurement target substance described above, but R ³ and R ⁴ are simultaneously hydrogen atoms. There is no such thing, and R ³ and R ⁴ may combine with each other to form a ring structure for detecting the substance to be measured. In this specification, the term “detection of a substance to be measured” includes detection by capturing ions and the like in addition to functional group conversion by chemical reaction with the substance to be measured. In this specification, the term “capture” refers to the case where R ³ and / or R ⁴ captures a metal ion or the like without causing a chemical change by chelation, or by chemical reaction with a measurement target substance. Including the case where the chemical structure of R ³ and / or R ⁴ changes, it should be interpreted in the broadest sense and should not be interpreted in any way restrictive.

Various substituents for capturing the substance to be measured have been proposed, and those skilled in the art can appropriately select depending on the type of the substance to be measured. For example, International Publication WO01 / 062755, JP-A-2004-315501 and the like can be referred to. In addition, Chapter 19 (calcium ion, magnesium ion, zinc ion, and other metal ions), Chapter 20 (pH indicator) of the catalog of Molecular Probes (Handbook of Fluorescent Probes and Research Chemicals, Tenth edition), In addition, substituents for capturing a substance to be measured described in Chapter 21 (sodium ion, potassium ion, chlorine ion, and other inorganic ions) can also be used. However, the substituent for capturing the substance to be measured is not limited to those described in the above publication. As a substituent for detecting an enzyme reaction substrate or enzyme reaction product, for example, when measuring sugar hydrolase activity, R ³ or R ⁴ is a residue of a sugar compound that becomes a substrate of the enzyme. Can be used. A functional group such as a hydroxy group or an amino group included in the sugar compound may be protected with an appropriate protecting group as necessary. All compounds having such protecting groups are also included in the scope of the present invention.

For example, in a fluorescent probe for measuring pH, one of R ³ and R ⁴ is a hydrogen atom, and the other is a C _1-18 alkyl group which may have a substituent, preferably a substituent. It may be a C _1-6 alkyl group which may have As the alkyl group, a linear alkyl group is preferable, and examples of the substituent that can be present on the optionally substituted C _1-18 alkyl group include an alkoxy group, an aryl group, and a halogen atom (fluorine atom, Any of chlorine atom, bromine atom and iodine atom), hydroxy group, amino group, carboxy group or ester thereof, sulfo group or ester thereof, phospho group or ester thereof, etc. The C _1-6 alkyl group that may be contained is preferably a carboxy-substituted C _1-6 alkyl group, and particularly preferably a 5-carboxypentyl group.

In the fluorescent probe used for pH measurement, R ³ and R ⁴ each independently have a C _1-18 alkyl group which may have a substituent, an aryl group which may have a substituent, or -(CH ₂ ) _m -N (R ²¹ ) (R ²² ), and at least one of R ³ and R ⁴ is-(CH ₂ ) _m -N (R ²¹ ) (R ²² ) (R ²¹ and R ²² Each independently represents an _optionally substituted C _1-6 alkyl group or an optionally substituted aryl group, or R ²¹ or R ²² is bonded to R ³ or R ⁴ , respectively. It is preferably an ethylene group, and m is 1 or 2. The C _1-18 alkyl group which may have a substituent represented by R ³ and R ⁴ is preferably a C _1-6 alkyl group which may have a substituent, particularly a methyl group. preferable. Examples of the substituent that can be present on the C _1-18 alkyl group which may have a substituent include an alkoxy group, an aryl group, and a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom). Or a hydroxy group, an amino group, a carboxy group or an ester thereof, or a sulfo group or an ester thereof. The C _1-18 alkyl group _optionally having a substituent represented by R ²¹ and R ²² is preferably a C _1-6 alkyl group _optionally having a substituent, particularly a methyl group, an ethyl group, Or a benzyl group is preferable. It is also preferred that R ²¹ or R ²² is bonded to R ³ or R ⁴ to form a C _1-3 alkylene group, and the alkylene group is an alkoxy group, aryl group, halogen atom (fluorine atom, chlorine atom, bromine). Any of an atom or an iodine atom), a hydroxyl group, an amino group, a carboxy group or an ester thereof, or a sulfo group or an ester thereof, and particularly preferably an ethylene group. . m is preferably 2. R ¹³ and R ¹⁴ in formula (AII) are the same as R ³ and R ⁴ in formula (AI).

Of these, fluorophore A is a group represented by AI,
(1) R ³ is a methyl group, and R ⁴ is a group represented by-(CH ₂ ) _m -N (R ²¹ ) (R ²² ),
When R ²¹ and R ²² are methyl groups and m is 2;
(2) R ³ is a methyl group, and R ⁴ is a group represented by-(CH ₂ ) _m -N (R ²¹ ) (R ²² ),
When R ²¹ and R ²² are ethyl groups and m is 2;
(3) R ³ is a group represented by-(CH ₂ ) _m -N (R ²¹ ) (R ²² ), m is 2, R ²¹ is a benzyl group, R ⁴ is R ²² An ethylene group bound to
(4) R ³ is a group represented by-(CH ₂ ) _m -N (R ²¹ ) (R ²² ), m is 2, R ²¹ is a methyl group, and R ⁴ is R ²² And an ethylene group bonded to
(5) R ³ is a group represented by-(CH ₂ ) _m -N (R ²¹ ) (R ²² ), m is 2, R ²¹ is a phenyl group, and R ⁴ is R ²² The case where it is the ethylene group couple | bonded with is preferable.

Further, for example, as a fluorescent probe for zinc ion measurement, R ³ and R ⁴ each independently represent a hydrogen atom, or the above formula (D) (wherein W ¹ , W ² , W ³ , and W ⁴ each independently represents a hydrogen atom, a 2-pyridylmethyl group, a 2-pyridylethyl group, a 2-methyl-6-pyridylmethyl group, or a 2-methyl-6-pyridylethyl group, W ¹ , W ² , At least one of the groups selected from the group consisting of W ³ and W ⁴ is a 2-pyridylmethyl group, a 2-pyridylethyl group, a 2-methyl-6-pyridylmethyl group, and a 2-methyl-6-pyridylethyl group A group selected from the group consisting of groups; p and q each independently represent 0 or 1). However, R ³ and R ⁴ are not both hydrogen atoms. In this case, p is 0, q is 1, W ⁴ is a hydrogen atom, and at least one of W ¹ and W ² is 2-pyridylmethyl group, 2-pyridylethyl group, 2-methyl A group selected from the group consisting of a -6-pyridylmethyl group and a 2-methyl-6-pyridylethyl group is preferable, and both W ¹ and W ² are particularly preferably a 2-pyridylmethyl group. Furthermore, p is 0, q is 0, and at least one of W ¹ and W ² is 2-pyridylmethyl group, 2-pyridylethyl group, 2-methyl-6-pyridylmethyl group, and 2-methyl A group selected from the group consisting of a -6-pyridylethyl group is also preferable.

In the formula (AI), R ¹ to R ⁴ may be groups independently embeddable in the cell membrane. In this case, only the pH change near the cell membrane can be measured using the compound of the present invention represented by the general formula (I) as a membrane-localized fluorescent probe. Groups that can be embedded in the cell membrane include linear or branched C _7-18 alkyl groups and phospholipids (eg, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, phosphatidylinositols, phosphatidylglycerols, cardiolipins, sphingos) Examples include myelins, ceramide phosphorylethanolamines, ceramide phosphorylglycerols, ceramide phosphorylglycerol phosphates, 1,2-dimyristoyl-1,2-deoxyphosphatidylcholines, plasmarogens, or phosphatidic acids. The fatty acid residue in the lipid is not particularly limited, and a phospholipid having one or two saturated or unsaturated fatty acid residues having about 12 to 20 carbon atoms can be used). ¹ to a substituent of R ⁴ And a group attached through a substituted group which may C _1-18 alkyl group are preferred.

Y ¹ and Y ² each independently represent —O—, —S—, —Se—, —CH═CH—, —C (R ⁵ ) (R ⁶ ) —, or —N (R ⁷ ) —, R ⁵ , R ⁶ and R ⁷ each independently represent a hydrogen atom or a C _1-6 alkyl group which may have a substituent. Y ¹ and Y ² are preferably —C (R ⁵ ) (R ⁶ ) —, and R ⁵ and R ⁶ are preferably methyl groups. Y ¹¹ and Y ¹² in Formula (AII) are the same as Y ¹ and Y ² in Formula (AI) above.

In the formula (AI), Z ¹ and Z ² each represent a nonmetallic atom group necessary for forming an optionally substituted benzo-fused ring or an optionally substituted naphtho-fused ring. More specifically, for example, Z ¹ and Z ² each form the following benzo-fused ring or the following naphtho-fused ring. The benzo-fused ring or naphtho-fused ring is further an alkoxy group, an aryl group, a halogen atom (any of a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), a hydroxy group, an amino group, a carboxy group or an ester thereof, or It may have a substituent such as a sulfo group or an ester thereof. Z ¹¹ and Z ¹² in Formula (AII) are the same as Z ¹ and Z ² in Formula (AI) above.

  In the formula (AI), the sum of n and n ′ is preferably 2, and both n and n ′ are particularly preferably 1. The fluorophore represented by the formula (AI) preferably has a maximum absorption wavelength in the region of 400 nm to 1300 nm, preferably 650 nm to 950 nm, more preferably 650 nm to 800 nm. In the fluorophore represented by the formula (AI) of the compound represented by the general formula (I) of the present invention, the person skilled in the art increases the excitation wavelength and the fluorescence wavelength as n or n ′ increases. It will be appreciated that when n or n ′ decreases, the excitation and fluorescence wavelengths decrease.

L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ and L ⁷ represent a substituted or unsubstituted methine group, which may be the same or different. When n is 2, the overlapping L ¹ and L ² may be the same or different, and when n ′ is 2, the overlapping L ⁶ and L ⁷ may be the same or different. The substituents of the methine groups represented by L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , and L ⁷ may be bonded to form a ring containing three consecutive methine groups. This ring may further form a condensed ring with a ring containing another methine group. As the partial structure constituted by L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , and L ⁷ , the following structures are particularly preferable.

  The fluorophore B is not particularly limited as long as it is a fluorophore serving as a donor or acceptor capable of inducing fluorescence resonance energy transfer with the fluorophore A represented by the formula (AI). A fluorophore can be selected. For example, in the formula (AI), when the sum of n and n ′ is 2, since the fluorophore A has a maximum excitation wavelength at about 650 nm to 800 nm, For example, a fluorophore having a fluorescent wavelength, for example, a cyanine fluorophore containing a tetramethylindodicarbocyanine derivative or a rhodamine derivative may be selected as appropriate. In this case, FRET efficiency to fluorophore A is measured by irradiating fluorophore B (when fluorophore B is a donor) with excitation light and measuring the fluorescence derived from each of fluorophore A and fluorophore B. Changes can be detected.

The length of the spacer represented by S can be appropriately selected within a range where FRET occurs between the fluorophore A and the fluorophore B. As the spacer, an alkyl group which may have a substituent represented by R ¹ to R ^{4 of the} fluorophore A represented by the formula (AI) in the general formula (I) may be used, and R ¹ Alternatively, an alkyl chain, a polyethylene glycol chain, or a polyamino acid may be introduced using a substituent of an alkyl group that may have a substituent represented by R ⁴ . Further, it is preferable that a structurally rigid group such as cyclohexane is contained in the spacer. In this case, the degree of freedom of the compound represented by the general formula (I) is decreased, and the donor and acceptor fluorophores are unlikely to associate with each other, and an effect of preventing formation of a non-fluorescent aggregate may be obtained. is there.

M ⁻ represents the number of counter ions necessary for charge neutralization. Examples of the counter ion include metal ions such as sodium ion, potassium ion, and magnesium ion, halogen ions such as quaternary ammonium and iodine ion, and amino acid ions such as glycine. For example, when a carboxy group, a sulfo group, etc. are present in the C _1-18 alkyl group represented by R ¹ and R ² of the fluorophore A represented by the formula (AI) in the general formula (I), or R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , Z ¹ , and Z ² include a carboxy group, a sulfo group, or a phospho group, etc., two or more counter ions as M ⁻ It may be necessary. Further, when one carboxy group or sulfo group or the like is present in one C _1-18 alkyl group represented by R ¹ and R ² of the fluorophore A represented by the formula (AI) in the general formula (I) When the positive charge on the quaternary nitrogen atom to which R ¹ is bound and the anion of the carboxy group or sulfo group form an intramolecular zitter ion, the counter ion necessary for charge neutralization is unnecessary. There is also.

  The compound of the present invention represented by the general formula (I) may have one or more asymmetric carbons. Thus, any optical isomer in optically pure form based on one or more asymmetric carbons, any mixture of optical isomers, racemates, diastereoisomers in pure form, diastereoisomerism Any mixture of bodies and the like is included within the scope of the present invention. Moreover, although the compound of this invention may exist as a hydrate or a solvate, it cannot be overemphasized that these substances are also included in the scope of the present invention.

In the general formula (I), the fluorophore A is represented by the formula (AI) (wherein R ¹ and R ² represent a methyl group and a 4-sulfobutyl group, and Y ¹ and Y ² represent —C (CH ₃ ) ₂ —. N and n ′ both represent 1, Z ¹ and Z ² represent an unsubstituted benzo-fused ring, and L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , and L ⁷ are unsubstituted It is preferable that the fluorophore B is a tetramethylindodicarbocyanine derivative. As the fluorescent probe for measuring pH, the following compounds can be mentioned as preferred compounds as the compounds represented by the general formula (I), but are not limited to these specific compounds.

The compound represented by the above general formula (I) can be produced, for example, by the method shown in the following scheme. In the examples of the present specification, the production methods of the representative compounds included in the general formula (I) are specifically shown. It will be understood by those skilled in the art that the compound represented by the above general formula (I) can be easily prepared by referring to the following schemes and specific examples.

The compound represented by the general formula (I) has a property of emitting strong fluorescence with excitation light in the near infrared region near 600 nm to 950 nm, which is excellent in biological tissue permeability, and has the formula (AI) The fluorophore A represented by (a) reacts with the substance to be measured in a substituent bonded to the methine chain such as L ¹ to change the electron donating ability; (b) a nitrogen atom directly connected to the cyanine fluorophore Has a property of causing a significant wavelength shift in the peak of the excitation spectrum according to the change in electron donating ability of the phosphor; and (c) FRET efficiency with the fluorophore B accompanying the wavelength shift of the peak of the excitation spectrum Has a property of substantially changing. For example, in the case of Compound 5, when the tricarbocyanine fluorophore is a pH-sensitive FRET acceptor, dicarbocyanine fluorophore is a FRET donor, and the nitrogen atom bound to the methine chain of the tricarbocyanine fluorophore is protonated. As the electron donating ability decreases, the peak of the excitation spectrum shifts from around 660 nm to a long wavelength. Due to this property, when irradiating light with an excitation wavelength of dicarbocyanine fluorophore (fluorescence with a fluorescence wavelength peak around 660 nm when 640 nm excitation light is applied), (1) when no protonation occurs In case of FRET from dicarbocyanine fluorophore to tricarbocyanine fluorophore efficiently, fluorescence from tricarbocyanine fluorophore (wavelength around 750 nm) is strongly observed, and (2) Protonation , The FRET from the dicarbocyanine fluorophore to the tricarbocyanine fluorophore does not occur efficiently, and the fluorescence from the dicarbocyanine fluorophore (wavelength around 660 nm) is strongly observed. In other words, compound 5 is dissolved in a test sample, excited with light at 640 nm, and the fluorescence intensity ratio between 660 nm and 750 nm at that time is measured to detect the pH by the ratio method. It becomes possible. Compound 5 is particularly useful as a pH fluorescent probe for measuring pH in living cells and living tissues, particularly in deep tissues, under physiological conditions.

  The ratio method in this specification is described in detail in Mason WT's book (Mason WT in Fluorescent and Luminescent Probes for Biological Activity, Second Edition, Edited by Mason WT, Academic Press). The specific example of the measuring method using the compound of this invention was also shown in the example. The term “measurement” used in this specification should be interpreted in the broadest sense including quantitative and qualitative. Further, the term “detection” used in this specification includes a concept of calculating a result using, for example, a measured value, for example, a concept of calculating a change in FRET efficiency.

  The method of using the fluorescent probe of the present invention is not particularly limited, and can be used in the same manner as conventionally known fluorescent probes. Usually, the above general formula (I) is used in an aqueous medium such as physiological saline or buffer, or a mixture of an aqueous medium such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, dimethylformamide and an aqueous medium. Dissolving a substance selected from the group consisting of a compound represented by the salt and a salt thereof, and adding this solution to an appropriate buffer containing cells and tissues, and (1) excellent permeability of an appropriately selected living tissue Or two-wavelength excitation-wavelength ratio measurement to measure each fluorescence intensity by exciting with two different wavelengths in the near-infrared region near 600 nm to 950 nm, or (2) transmission through living tissue One-wavelength excitation and two-wavelength fluorescence ratio measurement may be performed in which excitation is performed at one wavelength in the vicinity of 600 nm to 950 nm, which is excellent in performance, and fluorescence intensities of two different wavelengths selected as appropriate are measured. The pH fluorescent probe of the present invention may be used in the form of a composition in combination with an appropriate additive. For example, it can combine with additives, such as a buffer and a solubilizing agent.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples. (In the following examples, compound numbers correspond to the compound numbers in the above scheme.)
Example 1: Synthesis of compound 5
(A) Synthesis of Compound 1
2,3,3-Trimethyl-3H-indole (7.2 g, 45 mmol) and 3-iodopropionic acid (13 g, 63 mmol) were dissolved in o-dichlorobenzene. The mixture was heated and stirred at 110 ° C. for 15 hours under an argon atmosphere. The precipitate obtained by filtration was washed with diethyl ether and acetone to obtain Compound 1 (11 g).

(B) Synthesis of Compound 2 Malonaldehyde dianilide monohydrochloride (0.68 g, 2.6 mmol) and diisopropylethylamine (0.69 g, 5.3 mmol) were suspended in dichloromethane (6 mL). A mixed solution of acetic anhydride (0.36 g, 3.5 mmol) and dichloromethane (6 mL) was added dropwise thereto while stirring under ice cooling, and the mixture was further stirred for 1 hour under ice cooling (yellow reaction solution). Separately, Compound 1 and sodium acetate (0.85 g, 10 mmol) were dissolved in a mixed solution of acetonitrile (25 mL) and water (1 mL) and heated to reflux, and the previous yellow reaction solution was added dropwise thereto. After continuing to reflux for 1 hour, the mixture was allowed to cool to room temperature. The precipitate was collected by filtration and washed with acetonitrile, 5% aqueous hydrochloric acid solution and diethyl ether to obtain Compound 2 (0.68 g).

(C) Synthesis of Compound 3
IR-786 (Sigma, 0.50 g, 0.86 mmol) and 6-amino-n-caproic acid (0.46 g, 3.5 mmol) were dissolved in dimethylformamide (DMF, 30 mL) and kept at 80 ° C. for 3 hours under an argon atmosphere. Heated. After cooling to room temperature, the solvent was distilled off under reduced pressure. The residue was purified by column chromatography using NH silica gel to obtain Compound 3 (0.42 g).

(D) Synthesis of Compound 4
trans-1,4-cyclohexanediamine (0.19 g, 1.7 mmol) was dissolved in DMF (20 mL). Compound 3 (0.42 g, 0.61 mmol), 1-hydroxybenzotriazole monohydrate (HOBT · H ₂ O, 0.19 g, 1.3 mmol), O- (benzotriazol-1-yl) -N, N, A solution of N'-N'-tetramethyluronium hexafluorophosphate (HBTU, 0.47 g, 1.2 mmol) and diisopropylethylamine (85 mg, 0.66 mmol) in DMF (40 mL) was added dropwise with stirring at room temperature. . After stirring at room temperature for 3 hours under an argon atmosphere, the solvent was distilled off under reduced pressure. Purification by column chromatography using NH silica gel gave Compound 4 (0.35 g).

(E) Synthesis of Compound 5 Compound 4 (0.15 g, 0.21 mmol) was dissolved in DMF (20 mL). This solution was combined with DMF (20 mL) of Compound 2 (0.25 g, 0.40 mmol), HOBT · H ₂ O (94 mg, 0.61 mmol), HBTU (0.24 g, 0.62 mmol), diisopropylethylamine (85 mg, 0.66 mmol). ) The solution was added dropwise with stirring at room temperature. After stirring at room temperature for 3 hours under an argon atmosphere, the solvent was distilled off under reduced pressure. Purification by column chromatography using NH silica gel gave Compound 5 (35 mg).
¹ H-NMR (300 MHz, CD ₃ OD): δ 0.98-1.94 (m, 40 H), 2.03 (t, 2 H, J = 8.0 Hz), 2.41-2.52 (m, 8 H), 3.32 (s, 6 H), 3.40 (br, 2 H), 3.63 (t, 2 H, J = 6.8 Hz), 4.25 (br, 4 H), 5.65 (d, 2 H, J = 13.0 Hz), 6.15, (d , 1 H, J = 13.3 Hz), 6.33 (d, 1 H, J = 13.3 Hz), 6.51 (t, 1 H, J = 13.3 Hz), 6.94-7.36 (m, 16 H), 7.63 (d, 2H, J = 13.0 Hz), 8.11 (tt, 2 H, J = 13.2 Hz, 13.2 Hz)
HRMS (ESI ⁺ ) Calcd for [MH] ⁺ , 1154.7211, Found, 1154.7243

Example 2: Spectral change accompanying pH change of compound 5
Compound 5 in a 1: 1 mixed solution of 100 mmol / L phosphate buffer solution (pH 1.2, 2.1, 3.3, 3.9, 5.0, 6.0, 6.8) and acetonitrile (containing 0.1% DMSO or less as a co-solvent) The absorption spectrum and fluorescence spectrum (excitation wavelength: 640 nm) of were measured. The absorption spectrum is shown in FIG. 1 (A), and the fluorescence spectrum is shown in FIG. 1 (B). As the pH increases, the absorbance around 650 nm increases, indicating that the absorption spectrum changes. It can also be seen that the fluorescence spectrum decreases with increasing pH at around 660 nm and rises at around 750 nm. Accordingly, the pH change can be accurately measured using the ratio method based on the single wavelength excitation double wavelength fluorescence measurement using Compound 5. FIG. 1 (C) shows the fluorescence intensity ratio between 660 nm and 750 nm. Since the fluorescence intensity at 660 nm and 750 nm varies linearly from pH 4 to 6, pH in this range can be measured using Compound 5.

  By using the fluorescent probe provided by the present invention, the measurement target substance in the sample can be measured by the ratio method using the excitation wavelength / fluorescence wavelength in the near-infrared region which is excellent in biological tissue permeability.

Claims (5)

The following general formula (I):
Fluorophore A-S-Fluorophore B (I)
(However, fluorophore A and fluorophore B are fluorophores that emit fluorescence when irradiated with excitation light having a wavelength of 600 nm to 950 nm, respectively, and fluorophore A has a specific reaction with the analyte. Fluorescence properties change before and after, and S is a spacer that connects fluorophore A and fluorophore B), and fluorophore A and fluorescence before and after specific reaction with the analyte A fluorescent probe comprising a compound that produces a substantial change in fluorescence resonance energy efficiency with group B, wherein fluorophore A has the following formula (AII):

[Wherein, R ¹¹ and R ¹² each independently represent a C _1-6 alkyl group; R ¹³ and R ¹⁴ each independently represent a hydrogen atom, or a substituent for detecting a substance to be measured. However, R ¹³ and R ¹⁴ are not simultaneously hydrogen atoms, and R ¹³ and R ¹⁴ may combine with each other to form a ring structure for detecting the measurement target substance; Y ¹¹ and Y ¹² are Each independently represents —C (R ¹⁵ ) (R ¹⁶ ) — (wherein R ¹⁵ and R ¹⁶ each independently represents a hydrogen atom or a C _1-6 alkyl group); Z ¹¹ and Z ¹² each independently It represents a non-metallic atomic group necessary to form a good benzo condensed ring which may have a substituent; M ^'- represents a counter ion in a number required for neutralization of electric charge; provided R ¹³ and R the combination of ^14, a group maximum excitation wavelength of the fluorophores a represented by formula (AII) before and after the detection of the analyte is represented by a combination varying, firefly Fluorescent probe Dan B is tetramethyl indodicarbocyanine derivative or rhodamine derivative. The fluorescent probe according to claim 1 , wherein the substance to be measured is proton, metal ion, reactive oxygen species, enzyme reaction substrate or enzyme reaction product. The fluorescent probe according to claim 1 , which is a fluorescent probe for pH measurement, comprising the following compound.

A method for measuring fluorescence of a substance to be measured, comprising the following steps: (a) a step of bringing a compound represented by the general formula (I) according to claim 1 into contact with a substance to be measured; and (b) the above step A method comprising a step of measuring a fluorescence intensity of the compound represented by the general formula (I) after the contact in (a) and detecting a change in FRET efficiency due to the contact with the measurement target substance. The method according to claim 4, wherein the compound represented by the general formula (I) according to claim 1 is the compound according to claim 3.

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